The invention relates generally to the field of modulating the activity of the apoptotic protease-activating factor 1 (Apaf-1), which is an essential component of the apoptotic mechanism in mammalian cells.
Programmed cell death, or apoptosis, is essential to the development and homeostasis of metazoans (Danial et al., 2004, Cell 116:205-219; Horvitz, 2003, Chembiochem. 4:697-711). Abnormal inhibition of apoptosis is a hallmark of cancer and autoimmune diseases, and excessive activation of cell death is implicated in neuro-degenerative and other disorders (Hanahan et al., 2000, Cell 100:57-70; Yuan et al., 2000, Nature 407:802-809; Green et al., 2002, Cancer Cell 1: 19-30). Caspases, named after cysteine proteases that cleave after an aspartate residue in their substrates, are central components of the apoptotic response. The conserved mechanism of apoptosis across species involves a cascade of sequential activation of initiator and effector caspases (Riedl et al., 2004, Nature Rev. Mol. Cell. Biol. 5:897-907).
The caspase activation cascade downstream of mitochondria is controlled by Apaf-1, which is responsible for the activation of the initiator caspase-9 and subsequent activation of effector caspases-3 and -7 (Zou et al., 1997, Cell 90:405-413; Li et al., 1997, Cell 91:479-489). Apaf-1 has an essential role in the regulation of programmed cell death in mammalian development and in oncogene- and p53-dependent apoptosis (Cecconi et al., 1998, Cell 94:727-737; Yoshida et al., 1998, Cell 94:739-750; Soengas et al., 1999, Science 284:156-159; Fearnhead et al., 1998, Proc. Natl. Acad. Sci. 95:13664-1366). The importance of Apaf-1-mediated apoptosis is manifested by the observation that Apaf-1 is frequently inactivated in cancers such as malignant melanoma (Soengas et al., 2001, Nature 409:207-211).
In response to a wide range of intrinsic cell death stimuli, Apaf-1 interacts with cytosolic cytochrome c that is released from mitochondria and, in the presence of dATP or ATP, forms an oligomeric complex dubbed the apoptosome (Li et al., 1997, Cell 91:479-489; Zou et al., 1999, J. Biol. Chem. 274:11549-11556; Saleh et al., 1999, J. Biol. Chem. 274:17941-17945; Hu et al., 1999, EMBO J. 18:3586-3595). The mechanistic role of ATP/dATP-binding to Apaf-1 is unknown, although it is essential to the formation of the apoptosome (Li et al., 1997, Cell 91:479-489; Zou et al., 1999, J. Biol. Chem. 274:11549-11556; Saleh et al., 1999, J. Biol. Chem. 274:17941-17945; Hu et al., 1999, EMBO J. 18:3586-3595; Jiang et al., 2000, J. Biol. Chem. 275:31199-31203). The apoptosome, in turn, recruits and activates procaspase-9 and forms a holoenzyme with the processed caspase-9 (Rodriguez et al., 1999, Genes Dev. 13:3179-3184). In Drosophila, the Apaf-1 orthologue Dark (also known as Dapaf-1 and Hac-1; Rodriguez et al., 1999, Nat. Cell Biol. 1:272-279; Kanuka et al., 1999, Mol. Cell 4:757-769; Zhou et al., 1999, Mol. Cell 4:745-755) is critically important for activation of the initiator caspase Dronc (a caspase-9 orthologue). In C. elegans, CED-4 exhibits significant sequence homology to Apaf-1 and is indispensable for the activation of CED-3 (Zou et al., 1997, Cell 90:405-413; Yuan et al., 1992, Development 116:309-320), the only apoptotic caspase in worms.
Apaf-1 is a 140-kilodalton, multi-domain protein, consisting of an N-terminal caspase recruitment domain (CARD), a central nucleotide-binding domain, and 12-13 repeats of the WD40 domain at the C-terminal half. The WD40 repeats are thought to be responsible for binding to cytochrome c and are believed to have a regulatory role in Apaf-1 function, because the removal of the WD40 repeats resulted in a constitutively active Apaf-1 protein that activated caspase-9 in a cytochrome c-independent manner (Hu et al., 1998, J. Biol. Chem. 273:33489-33494; Srinivasula et al., 1998, Mol. Cell 1:949-957). However, the underlying molecular mechanisms of how Apaf-1 interacts with ATP/dATP during formation of the apoptosome and activation of caspase-9 were not previously understood.
Apaf-1 is a representative member of the nucleotide-binding oligomerization (NOD) family of proteins that, in addition to Dark and CED-4, also include Ipaf, Nod1, Nod2, and a large family of disease-resistant proteins in plants (Inohara et al., 2001, Oncogene 20:6473-6481; Poyet et al., 2001, J. Biol. Chem. 276:28309-28313; Inohara et al., 1999, J. Biol. Chem. 274:14560-14567; Ogura et al., 2001, J. Biol. Chem. 276:4812-4818; Dangl et al., 2001, Nature 411:826-833). The hallmark of these proteins is the central NOD domain flanked by an N-terminal homotypic interaction motif and a C-terminal ligand-sensing domain. The shared domain structure suggests conserved mechanisms of action. However, the lack of structural information on any member of the NOD family proteins severely restricts our understanding on the mechanisms of the NOD family of proteins.
Efforts to study Apaf-1 protein have been hampered by inability of others to generate significant quantities of the protein in a form sufficiently stable, soluble, and pure to allow such study. For instance, there is no published protocol that allows bacterial expression and purification of a soluble recombinant Apaf-1 fragment longer than 200 amino acids. In addition, there is no published protocol that allows the preparation of a soluble, stable, recombinant Apaf-1 fragment longer than 200 amino acids, except for the full-length Apaf-1 protein in baculovirus-infected insect cells.
As a consequence of the lack of availability of reasonable quantities of Apaf-1 protein for research studies, little work has been done to identify compounds which can modulate the activity of Apaf-1. Furthermore, there has been an absence of three-dimensional structure information for any fragment of Apaf-1 other than the soluble N-terminal CARD domain. Knowledge of the physical structure of Apaf-1 protein would significantly aid design and screening of compounds that can modulate the activity of Apaf-1.
The present invention overcomes prior limitations by providing a method of producing stable, soluble, pure, and active Apaf-1 protein. The invention includes a description of the three-dimensional structure of Apaf-1 and methods of screening compounds to assess their ability to modulate Apaf-1 activity.